During the analysis of samples under the microscope, the object often arises of observing the sample from different directions, i.e. at different orientation angles using an optical contrast method, for example using a fluorescence method. This can be advantageous for example when the excitation light and/or the detection light is developed differently for different orientations, i.e. in particular has different intensities, which can be caused for example by scattering processes in the sample. In order to receive a spatial impression of the sample, it has in addition become usual to analyse the sample acquisition by an optical sectioning method, for which an image stack of various parallel planes spaced apart along the detection direction D, the axis ZD, is acquired for each orientation angle. From this image stack, an image of the sample can then be generated which conveys a spatial impression. The sample can be moved, perpendicular to the detection direction D, in the directions XD and YD by a motorized positioning unit. In order to produce the different orientations of the sample, the latter is, as described above, supported rotatable about at least one axis of rotation R.
These so-called multiview acquisitions are also of interest in particular for examining larger samples using a SPIM method (selective/single-plane illumination microscopy). In this method, the sample is illuminated perpendicular to the detection direction D, thus in the XD-YD plane here, with a light sheet which can be generated statically or dynamically, the latter for example by point scanning. The observation can be carried out in wide field, with the result that a larger sample region can be captured without, however, the sample fading in a large volume. Not least for this reason, the significance of such SPIM methods has become much more important for the fluorescence microscopy analysis of samples, as can be learnt for example from the article “Light Sheet Fluorescence Microscopy: A Review” by Peter A. Santi, published in the Journal of Hystochemistry and Zytochemistry, vol. 59, pp. 129-138 from 2011. The main features of the method are also described for example in WO 2004/053558 A1.
Similarly to confocal laser scanning microscopy, SPIM methods, as wide-field observation methods, allow spatially extended objects to be acquired in the form of optical sections, wherein the advantages consist above all in the speed, the low level of fading of the sample as well as a broadened penetration depth. Unlike conventional fluorescence contrast methods in microscopy using reflected light or transmitted light methods—as examples there may be mentioned here epifluorescence, confocal laser scanning microscopy and multiphoton microscopy—the fluorophores in the sample are excited using laser light in the form of a light sheet or several light sheets. The light sheet can, as already indicated above, be generated by a scanning mechanism, but it can also be generated statically, for example with the aid of a cylindrical lens. As a rule, the sample is held on a positioning unit by a sample holder that can be moved in all spatial directions, in addition the sample holder also has an axis of rotation. The sample can also be supported in a gel, with the result that it cannot be moved compared with the positionable sample holder.
For multiview acquisitions, the sample is rotated and images are acquired from several directions, at several orientation angles. As most applications are to be found in the field of developmental biology, these acquisitions take place at several points in time, which can be distributed over a period of from several hours up to several days. Sometimes the first points in time are of particular interest above all and an effort is made to keep the period of sample preparation in the microscope as small as possible, in order not to miss any important development steps. For this reason, an automation of this type of acquisition is of interest, in particular also as regards the preparation for these acquisitions.
In the state of the art, the following steps are usually carried out for preparing for and carrying out multiview acquisitions.    1. A first orientation angle is set.    2. The positioning unit with the sample holder and the sample is set such that the sample or the sample region of interest moves into the image field region and moreover at least part of the sample is imaged in sharp definition, i.e. the focal plane lies in the sample.    3. In order to define the limits for the acquisition of the image stack in the ZD direction, a first z position is sought and stored, in which the sample region of interest is located directly in front of or behind the focal plane of the detection objective.    4. A second z position is stored when the positioning unit is set such that the sample region of interest is located directly behind or in front of the focal plane of the detection objective.    5. Both z positions are stored as initial or final parameters together with the orientation angle and the position of the sample in the XD-YD plane, with the result that the parameters for defining the image stack are known.    6. Then the next orientation angle is set and steps 2.-5. are repeated.    7. Steps 2-6 are repeated until the desired number of orientation angles has been reached.
At the end there is a set of parameters for defining the coordinates for the acquisition of image stacks along the detection direction D for each orientation angle.
A problem with this method is in particular that of finding the sample region of interest after setting a new orientation angle, in particular in the case of small image field sizes, or in the case of a large distance between the sample and the axis of rotation about which the sample is rotated. Precisely in the latter case, the sample region of interest in some cases also moves out of the region of the image field even in the case of very small angular movements, as the position of the sample changes, due to the rotation, in the direction of the axes XD and ZD relative to the focused image field centre, i.e. in relation to the detection coordinate system. The position of the sample holder, i.e. as a rule the motor position of a positioning unit to which the sample holder is attached, must be changed by the amounts Δx, Δz in order to bring the sample back into the focal centre and the image field centre. The reason is that the sample does not, as a rule, lie precisely on the axis of rotation R. It is thereby made much more difficult to prepare for and carry out multiview acquisitions.
An alternative is to observe the sample at a smaller magnification first for preparing for a new image stack acquisition at another orientation angle, with the result that during the rotation the sample no longer moves out of the image field so quickly in the plane perpendicular to the axis of rotation. This can be performed for example by changing the objective on the revolving objective holder, or else by a zoom system independent of the objective and integrated in the illumination beam path. However, this means that an additional work step has to be carried out. The remaining work steps, such as setting each orientation angle and defining the boundary values on the ZD axis for the stack acquisition, furthermore also have to be carried out.
The automated tracking of the sample table or the sample holder during the rotation offers a certain facilitation, which is, however, only possible when the position of the axis of rotation R, i.e. the position of the sample coordinate system in relation to the position of the detection coordinate system, is known. The entire sample holder and, with it, the sample coordinate system can then be shifted in the detection coordinate system such that the sample remains in the image field, thus the sample holder is tracked translationally. If the position of the axis of rotation in these so-called motor coordinates—i.e. also in the detection coordinate system—is known, the tracking can be determined for each angular movement with the aid of simple transformation matrices, a pure rotation matrix in the most favourable case, and set correspondingly. Even in this case, however, the manual search for the optimal parameters for limiting the image stack in the ZD direction and for setting each individual angle is not dispensed with.